Gonadotropin-releasing hormone receptor antagonists and methods relating thereto

ABSTRACT

GnRH receptor antagonists are disclosed that have utility in the treatment of a variety of sex-hormone related conditions in both men and women. The compounds of this invention have the structure:  
                 
 
wherein R 1a , R 1b , R 1c , R 2a , R 2b , R 3 , R 4 , R 5 , R 6  and X are as defined herein, including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof. Also disclosed are compositions containing a compound of this invention in combination with a pharmaceutically acceptable carrier, as well as methods relating to the use thereof for antagonizing gonadotropin-releasing hormone in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 60/485,434 filed Jul. 7, 2003, which application isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

Partial funding of the work described herein was provided by the U.S.Government under Grant No. 1-R₄₃-HD38625 and 2R₄₄-HD38625-02 provided bythe National Institutes of Health. The U.S. Government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to gonadotropin-releasing hormone(GnRH) receptor antagonists, and to methods of treating disorders byadministration of such antagonists to a warm-blooded animal in needthereof.

2. Description of the Related Art

Gonadotropin-releasing hormone (GnRH), also known as luteinizinghormone-releasing hormone (LHRH), is a decapeptide(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) that plays an importantrole in human reproduction. GnRH is released from the hypothalamus andacts on the pituitary gland to stimulate the biosynthesis and release ofluteinizing hormone (LH) and follicle-stimulating hormone (FSH). LHreleased from the pituitary gland is responsible for the regulation ofgonadal steroid production in both males and females, while FSHregulates spermatogenesis in males and follicular development infemales.

Due to its biological importance, synthetic antagonists and agonists toGnRH have been the focus of considerable attention, particularly in thecontext of prostate cancer, breast cancer, endometriosis, uterineleiomyoma (fibroids), ovarian cancer, prostatic hyperplasia, assistedreproductive therapy, and precocious puberty (The Lancet 358:1793-1803,2001; Mol. Cell. Endo. 166:9-14, 2000). For example, peptidic GnRHagonists, such as leuprorelin(pGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHEt), have been used to treatsuch conditions. Such agonists appear to function by binding to the GnRHreceptor in the pituitary gonadotropins, thereby inducing the synthesisand release of gonadotropins. Chronic administration of GnRH agonistsdepletes gonadotropins and subsequently down-regulates the receptor,resulting in suppression of steroidal hormones after some period of time(e.g., on the order of 2-3 weeks following initiation of chronicadministration).

In contrast, GnRH antagonists are believed to suppress gonadotropinsfrom the onset, and thus have received the most attention over the pasttwo decades. To date, some of the primary obstacles to the clinical useof such antagonists have been their relatively low bioavailability andadverse side effects caused by histamine release. However, severalpeptidic antagonists with low histamine release properties have beenreported, although they still must be delivered via sustained deliveryroutes (such as subcutaneous injection or intranasal spray) due tolimited bioavailability.

In view of the limitations associated with peptidic GnRH antagonists, anumber of nonpeptidic compounds have been proposed. For example, Cho etal. (J. Med. Chem. 41:4190-4195, 1998) disclosesthieno[2,3-b]pyridin-4-ones for use as GnRH receptor antagonists; U.S.Pat. Nos. 5,780,437 and 5,849,764 teach substituted indoles as GnRHreceptor antagonists (as do published PCTs WO 97/21704, 98/55479,98/55470, 98/55116, 98/55119, 97/21707, 97/21703 and 97/21435);published PCT WO 96/38438 discloses tricyclic diazepines as GnRHreceptor antagonists; published PCTs WO97/14682, 97/14697 and 99/09033disclose quinoline and thienopyridine derivatives as GnRH antagonists;published PCTs WO 97/44037, 97/44041, 97/44321 and 97/44339 teachsubstituted quinolin-2-ones as GnRH receptor antagonists; and publishedPCT WO 99/33831 discloses certain phenyl-substituted fusednitrogen-containing bicyclic compounds as GnRH receptor antagonists.Recently published PCTs WO 02/066459 and WO 02/11732 disclose the use ofindole derivatives and novel bicyclic and tricyclic pyrrolidinederivatives as GnRH antagonists, respectively. Other recently publishedPCTs which disclose compounds and their use as GnRH antagonists includeWO 00/69859, WO 01/29044, WO 01/55119, WO 03/013528, WO 03/011870, WO03/011841, WO 03/011839 and WO 03/011293.

While significant strides have been made in this field, there remains aneed in the art for effective small molecule GnRH receptor antagonists.There is also a need for pharmaceutical compositions containing suchGnRH receptor antagonists, as well as methods relating to the usethereof to treat, for example, sex-hormone related conditions. Thepresent invention fulfills these needs, and provides other relatedadvantages.

BRIEF SUMMARY OF THE INVENTION

In brief, this invention is generally directed to gonadotropin-releasinghormone (GnRH) receptor antagonists, as well as to methods for theirpreparation and use, and to pharmaceutical compositions containing thesame. More specifically, the GnRH receptor antagonists of this inventionare compounds having the following general structure (I):

including stereoisomers, prodrugs and pharmaceutically acceptable saltsthereof, wherein R_(1a), R_(1b), R_(1c), R_(2a), R_(2b), R₃, R₄, R₅, R₆and X are as defined below.

The GnRH receptor antagonists of this invention have utility over a widerange of therapeutic applications, and may be used to treat a variety ofsex-hormone related conditions in both men and women, as well as amammal in general (also referred to herein as a “subject”). For example,such conditions include endometriosis, uterine fibroids, polycysticovarian disease, hirsutism, precocious puberty, gonadalsteroid-dependent neoplasia such as cancers of the prostate, breast andovary, gonadotrophe pituitary adenomas, sleep apnea, irritable bowelsyndrome, premenstrual syndrome, benign prostatic hypertrophy,contraception and infertility (e.g., assisted reproductive therapy suchas in vitro fertilization). The compounds of this invention are alsouseful as an adjunct to treatment of growth hormone deficiency and shortstature, and for the treatment of systemic lupus erythematosis. Thecompounds are also useful in combination with androgens, estrogens,progesterones, and antiestrogens and antiprogestogens for the treatmentof endometriosis, fibroids, and in contraception, as well as incombination with an angiotensin-converting enzyme inhibitor, anangiotensin II-receptor antagonist, or a renin inhibitor for thetreatment of uterine fibroids. In addition, the compounds may be used incombination with bisphosphonates and other agents for the treatmentand/or prevention of disturbances of calcium, phosphate and bonemetabolism, and in combination with estrogens, progesterones and/orandrogens for the prevention or treatment of bone loss or hypogonadalsymptoms such as hot flashes during therapy with a GnRH antagonist.

The compounds of the present invention, in addition to their GnRHreceptor antagonist activity, possess a reduced interaction with themajor metabolic enzymes in the liver, namely the Cytochrome P450enzymes. This family of enzymes, which includes the subtypes CYP2D6 andCYP3A4, is responsible for the metabolism of drugs and toxins leading totheir disposition from the body. Inhibition of these enzymes can lead tolife-threatening conditions where the enzyme is not able to perform thisfunction.

The methods of this invention include administering an effective amountof a GnRH receptor antagonist, preferably in the form of apharmaceutical composition, to a mammal in need thereof. Thus, in stilla further embodiment, pharmaceutical compositions are disclosedcontaining one or more GnRH receptor antagonists of this invention incombination with a pharmaceutically acceptable carrier and/or diluent.

These and other aspects of the invention will be apparent upon referenceto the following detailed description. To this end, various referencesare set forth herein which describe in more detail certain backgroundinformation, procedures, compounds and/or compositions, and are eachhereby incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention is directed generally tocompounds useful as gonadotropin-releasing hormone (GnRH) receptorantagonists. The compounds of this invention have the followingstructure (I):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,

-   -   wherein:        -   R_(1a), R_(1b) and R_(1c) are the same or different and            independently hydrogen, halogen, C₁₋₄alkyl, hydroxy or            alkoxy, or R_(1a) and R_(1b) taken together form —OCH₂O— or            —OCH₂CH₂—;        -   R_(2a) and R_(2b) are the same or different and            independently hydrogen, halogen, trifluoromethyl, cyano or            —SO₂CH₃;        -   R₃ is hydrogen or methyl;        -   R₄ is phenyl or C₃₋₇alkyl;        -   R₅ is hydrogen or C₁₋₄alkyl;        -   R₆ is —COOH or an acid isostere; and        -   X is C₁₋₆alkanediyl optionally substituted with from 1 to 3            C₁₋₆alkyl groups.

As used herein, the above terms have the following meaning:

“C₁₋₆alkyl” means a straight chain or branched, noncyclic or cyclic,unsaturated or saturated aliphatic hydrocarbon containing from 1 to 6carbon atoms. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; whilesaturated branched alkyls include isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, and the like. Representative saturated cyclicalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and thelike; while unsaturated cyclic alkyls include cyclopentenyl andcyclohexenyl, and the like. Unsaturated alkyls contain at least onedouble or triple bond between adjacent carbon atoms (referred to as an“alkenyl” or “alkynyl”, respectively). Representative straight chain andbranched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; whilerepresentative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,3-methyl-1-butynyl, and the like.

“C₁₋₄alkyl” means a straight chain or branched, noncyclic or cyclichydrocarbon containing from 1 to 4 carbon atoms. Representative straightchain alkyls include methyl, ethyl, n-propyl, n-butyl, and the like;branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, andthe like; while cyclic alkyls include cyclopropyl and the like.

“C₃₋₇alkyl” means a straight chain or branched, noncyclic or cyclichydrocarbon containing from 3 to 7 carbon atoms. Representative straightchain alkyls include n-propyl, n-butyl, n-hexyl, and the like; whilebranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative cyclic alkyls includecyclopropyl, cyclopentyl, cyclohexyl, and the like.

“C₁₋₆alkanediyl” means a divalent C₁₋₆alkyl from which two hydrogenatoms are taken from the same carbon atom or from difference carbonatoms, such as —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, and the like.

“Halogen” means fluoro, chloro, bromo or iodo, typically fluoro andchloro.

“Hydroxy” means —OH.

“Alkoxy” means —O-(C₁₋₆alkyl).

“Cyano” means —CN.

“Acid isostere” means an moiety that exhibits properties similar tocarboxylic acid, and which has a pKa of less than 8 and preferably lessthan 7. Representative acid isosteres include tetrazole,3H-[1,3,4]oxadiazol-2-one, [1,2,4]oxadiazol-3-one,1,2-dihydro-[1,2,4]triazol-3-one, 2H-[1,2,4]oxadiazol-5-one, triazolesubstituted with a sulfonyl or sulfoxide group, imidazole substitutedwith a sulfonyl or sulfoxide group, [1,2,4]-oxadiazolidine-3,5-dione,[1,2,4]-thiadiazolidine-3,5-dione, imidazolidine-2,4-dione,imidazolidine-2,4,5-trione, pyrrolidine-2,5-dione andpyrrolidine-2,3,5-trione. Acid isosteres also include—C(═O)NHSO₂NR_(a)R_(b), —C(═O)NHSO₂R_(b), —C(═O)NHC(═O)NR_(a)R_(b) and—C(═O)NHC(═O)R_(b), where R_(a) is hydrogen or C₁₋₄alkyl and R_(b) isC₁₋₄alkyl.

In one embodiment, R₄ is phenyl and representative GnRH antagonists ofthe present invention include compounds having the following structure(II).

In another embodiment, R₄ is C₃₋₇alkyl and representative GnRHantagonists of the present invention include compounds having thefollowing structure (III).

In more specific embodiments of structure (III), C₃₋₇alkyl is a straightchain or branched C₃₋₇alkyl such as isobutyl as represented by structure(IV), or is a cyclic C₃₋₇alkyl such as cyclohexyl as represented bystructure (V):

In another embodiment, R_(1a), R_(1b) and R_(1c) are hydrogen, alkoxyand halogen, respectively. A representative substitution patternincludes 2-halo-3-alkoxy-phenyl. Representative alkoxy groups includemethoxy and ethoxy, while representative halogen moieties include fluoroand chloro.

In an alternative embodiment, R_(1a) and R_(1b) taken together form—OCH₂O—, such as 3,4-methylene-dioxy.

In a further embodiment, R_(2a) and R_(2b) are hydrogen,trifluoromethyl, halogen or —SO₂CH₃. A representative substitutionpattern includes R_(2a) as halogen at the 2-position and R_(2b) ashydrogen, trifluoromethyl, halogen or —SO₂CH₃ at the 6-position.

Further embodiments include those wherein R₅ is H or methyl; R₆ is—COOH, and/or X is —CH₂CH₂—, —CH₂CH₂CH₂— or —CH₂CH₂CH₂CH₂—.

The compounds of the present invention may be prepared by known organicsynthesis techniques, including the methods described in more detail inthe Examples. In general, the compounds of structure (I) above may bemade by the following reaction schemes, wherein all substituents are asdefined above unless indicated otherwise.

An appropriately substituted benzonitrile may be reduced using anappropriate reagent such as borane in THF to the corresponding amine andthen forms urea 1. Cyclization with a reagent such as diketene givescompound 2 which may be brominated with bromine in acetic acid,N-bromosuccinimide or other brominating agent to give compound 3.Alkylation gives compound 4 and Suzuki condensation with a boronic acidor boronic acid ester gives compound 5. Deprotection of the protectedamine using a typical reagent (such as trifluoroacetic acid in methylenechloride in the case of a BOC group) gives compound 6 which may bealkylated or condensed with an aldehyde via reductive aminationconditions to give a compound of formula 7. It is possible to alter theorder of the various reductive amination, alkylation, bromination andSuzuki condensation steps to give compounds of the present invention.

In a variation of Scheme 1, compound 4 undergoes deprotection to givecompound 8 which under Suzuki conditions gives compound 6. The —X—R₆group may be added by alkylation, reductive amination or other reactionto give compound 7.

Substituted phenylacetic acid ester 9 (made form the corresponding acidor purchased) and reagent such as dimethylformamide dimethylacetal arecondensed to give 10. Cyclization with urea gives a compound of formula11. Alkylation using, for example, a substituted benzyl bromide gives 12which may be alkylated with an appropriate alkyl halide, undergo aMitsonobu coupling reaction with an appropriate alcohol, or react with amesylate or sulfonate to give 5.

The compounds of the present invention may generally be utilized as thefree acid or free base. Alternatively, the compounds of this inventionmay be used in the form of acid or base addition salts. Acid additionsalts of the free amino compounds of the present invention may beprepared by methods well known in the art, and may be formed fromorganic and inorganic acids. Suitable organic acids include maleic,fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic,trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric,gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic,glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acidsinclude hydrochloric, hydrobromic, sulfuric, phosphoric, and nitricacids. Base addition salts included those salts that form with thecarboxylate anion and include salts formed with organic and inorganiccations such as those chosen from the alkali and alkaline earth metals(for example, lithium, sodium, potassium, magnesium, barium andcalcium), as well as the ammonium ion and substituted derivativesthereof (for example, dibenzylammonium, benzylammonium,2-hydroxyethylammonium, and the like). Thus, the term “pharmaceuticallyacceptable salt” of structure (I) is intended to encompass any and allacceptable salt forms.

In addition, prodrugs are also included within the context of thisinvention. Prodrugs are any covalently bonded carriers that release acompound of structure (I) in vivo when such prodrug is administered to apatient. Prodrugs are generally prepared by modifying functional groupsin a way such that the modification is cleaved, either by routinemanipulation or in vivo, yielding the parent compound. Prodrugs include,for example, compounds of this invention wherein hydroxy, amine orsulfhydryl groups are bonded to any group that, when administered to apatient, cleaves to form the hydroxy, amine or sulfhydryl groups. Thus,representative examples of prodrugs include (but are not limited to)acetate, formate and benzoate derivatives of alcohol and aminefunctional groups of the compounds of structure (I). Further, in thecase of a carboxylic acid (—COOH), esters may be employed, such asmethyl esters, ethyl esters, and the like.

With regard to stereoisomers, the compounds of structure (I) may havechiral centers and may occur as racemates, racemic mixtures and asindividual enantiomers or diastereomers. All such isomeric forms areincluded within the present invention, including mixtures thereof.Furthermore, some of the crystalline forms of the compounds of structure(I) may exist as polymorphs, which are included in the presentinvention. In addition, some of the compounds of structure (I) may alsoform solvates with water or other organic solvents. Such solvates aresimilarly included within the scope of this invention.

The effectiveness of a compound as a GnRH receptor antagonist may bedetermined by various assay techniques. Assay techniques well known inthe field include the use of cultured pituitary cells for measuring GnRHactivity (Vale et al., Endocrinology 91:562-572, 1972) and themeasurement of radioligand binding to rat pituitary membranes (Perrin etal., Mol. Pharmacol. 23:44-51, 1983) or to membranes from cellsexpressing cloned receptors as described below. Other assay techniquesinclude (but are not limited to) measurement of the effects of GnRHreceptor antagonists on the inhibition of GnRH-stimulated calcium flux,modulation of phosphoinositol hydrolysis, and the circulatingconcentrations of gonadotropins in the castrate animal. Descriptions ofthese techniques, the synthesis of radiolabeled ligand, the employmentof radiolabeled ligand in radioimmunoassay, and the measurement of theeffectiveness of a compound as a GnRH receptor antagonist follow.

Inhibition of GnRH Stimulated LH Release

Suitable GnRH antagonists are capable of inhibiting the specific bindingof GnRH to its receptor and antagonizing activities associated withGnRH. For example, inhibition of GnRH stimulated LH release in immaturerats may be measured according to the method of Vilchez-Martinez(Endocrinology 96:1130-1134, 1975). Briefly, twenty-five day old maleSpraque-Dawley rats are administered an GnRH antagonist in saline orother suitable formulation by oral gavage, subcutaneous injection, orintravenous injection. This is followed by subcutaneous injection of 200ng GnRH in 0.2 ml saline. Thirty minutes after the last injection, theanimals are decapitated and trunk blood is collected. Aftercentrifugation, the separated plasma is stored at −20° C. untildetermination of the concentrations of LH and/or FSH by radioimmunoassay(see below.)

Rat Anterior Pituitary Cell Culture Assay of GnRH Antagonists

Anterior pituitary glands are collected from 7-week-old femaleSprague-Dawley rats and the harvested glands are digested withcollagenase in a dispersion flask for 1.5 hr at 37° C. After collagenasedigestion, the glands are further digested with neuraminidase for 9 minat 37° C. The digested tissue is then washed with 0.1% BSA/McCoy's 5Amedium, and the washed cells are suspended in 3% FBS/0.1 BSA/McCoy's 5Amedium and plated onto 96-well tissue culture plates at a cell densityof 40,000 cells per well in 200 μl medium. The cells are then incubatedat 37° C. for 3 days. For assay of an GnRH antagonist, the incubatedcells are first washed with 0.1% BSA/McCoy's 5A medium once, followed byaddition of the test sample plus 1 nM GnRH in 200 μl 0.1% BSA/McCoy's 5Amedium in triplicate wells. Each sample is assayed at 5-dose levels togenerate a dose-response curve for determination of the potency on theinhibition of GnRH stimulated LH and/or FSH release. After 4-hrincubation at 37° C., the medium is harvested and the level of LH and/orFSH secreted into the medium is determined by RIA.

Membrane Binding Assays 1

Cells stably, or transiently, transfected with GnRH receptor expressionvectors are harvested, resuspended in 5% sucrose and homogenized using apolytron homogenizer (2×15 sec). Nucleii are removed by centrifugation(3000×g for 5 min.), and the supernatant is centrifuged (20,000×g for 30min, 4° C.) to collect the membrane fraction. The final membranepreparation is resuspended in binding buffer (10 mM Hepes (pH 7.5), 150mM NaCl, and 0.1% BSA) and stored at −70° C. Binding reactions areperformed in a Millipore MultiScreen 96-well filtration plate assemblywith polyethylenimine coated GF/C membranes. The reaction is initiatedby adding membranes (40 μg protein in 130 ul binding buffer) to 50 μl of[¹²⁵I]-labeled GnRH peptide (˜100,000 cpm) and 20 μl of competitor atvarying concentrations. The reaction is terminated after 90 minutes byapplication of vacuum and washing (2×) with phosphate buffered saline.Bound radioactivity is measured using 96-well scintillation counting(Packard Topcount) or by removing the filters from the plate and directgamma counting. K_(i) values are calculated from competition bindingdata using non-linear least squares regression using the Prism softwarepackage (GraphPad Software).

Membrane Binding Assays 2

For additional membrane binding assays, stably transfected HEK293 cellsare harvested by striking tissue culture flasks against a firm surfaceand collected by centrifugation at 1000×g for 5 minutes. Cell pelletsare resuspended in 5% sucrose and homogenized using a polytronhomogenizer for two 15 second homogenization steps. Cell homogenates arethen centrifuged for 5 minutes at 3000×g to remove nuclei, and thesupernatant is subsequently centrifuged for 30 minutes at 44,000×g tocollect the membrane fraction. The membrane pellet is resuspended inGnRH binding buffer (10 mM HEPES, pH 7.5, 150 mM NaCl and 0.1% BSA) andaliquots are immediately snap-frozen in liquid nitrogen and stored at−80° C. Protein content of the membrane suspension is determined usingthe Bio-Rad protein assay kit (Bio-Rad, Hercules, Calif.).

Competitive radioligand binding assays with membrane preparations areperformed in Millipore 96-well filtration plates with GF/C membranefilters which are precoated with 200 μl of 0.1% polyethylenimine (Sigma,St. Louis. MO). Prior to use, the plates are washed 3× with phosphatebuffered saline solution. Membrane fraction in GnRH binding buffer (130μl containing 25 μg protein for human and macaque receptors or 12 μg forrat receptors) are added to wells together with 20 μl of competingligand at varying concentrations. The binding reaction is initiated byaddition of radioligand (0.1 nM in 50 μl GnRH binding buffer.) Thereaction is allowed to proceed for 90 min on a platform shaker at roomtemperature and then terminated by placing assay plate on a Milliporevacuum manifold (Millipore, Bedford, Mass.), aspirating the solvent, andwashing wells twice with 200 μl ice cold phosphate buffered saline(PBS). Filters in the wells are removed and counted in a gamma counter.K_(i) values are calculated from each competition binding curves usingnon-linear least square regression and corrected for radioligandconcentration using the Cheng-Prusoff equation (Prism, GraphPadSoftware, San Diego, Calif.) assuming a radioligand affinity of 0.5 nM.Mean K_(i) values are calculated from the antilog of the mean of thepK_(i) values for each receptor ligand pair.

Membrane Binding Assays 3

Stably transfected human GnRH receptor RBL cells are grown toconfluence. The medium is removed and the cell monolayer is washed oncewith DPBS. A solution of 0.5 mM EDTA/PBS (Ca⁺⁺ Mg⁺⁺ free) is added tothe plate which is then incubated at 37° C. for 10 min. Cells aredislodged by gentle rapping of the flasks. The cells are collected andpelleted by centrifugation at 800 g for 10 min at 4° C. The cell pelletis then resuspended in buffer [DPBS (1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, 2.7mM KCl, and 138 mM NaCl) supplemented with 10 mM MgCl₂, 2 mM EGTA,pH=7.4 with NaOH]. Cell lysis is then performed using a pressure celland applying N₂ at a pressure of 900 psi for 30 min at 4° C. Unbrokencells and larger debris are removed by centrifugation at 1200 g for 10min at 4° C. The cell membrane supernatant is then centrifuged at 45,000g and the resulting membrane pellet is resuspended in assay buffer andhomogenized on ice using a tissue homogenizer. Protein concentrationsare determined using the Coomassie Plus Protein Reagent kit (Pierce,Rockford, Ill.) using bovine serum albumin as a standard. The pelletsare aliquoted and stored at −80° C. until use. Titration analysis usinga range of protein concentrations determined the optimal proteinconcentration to be 15 μg per well final concentration.

UniFilter GF/C filter plates (Perkin Elmer, Boston Mass.) are pretreatedwith a solution of 0.5% polyethyleneimine in distilled water for 30minutes. Filters are pre-rinsed with 200 μl per well of PBS, 1% BSA(Fraction V) and 0.01% Tween-20, pH=7.4) using a cell harvester(UniFilter-96 Filtermate; Packard). Membranes are harvested by rapidvacuum filtration and washed 3 times with 250 μl of ice-cold buffer(PBS, 0.01% Tween-20, pH=7.4). Plates are air dried, 50 μl scintillationfluid (Microscint 20; Packard) is added, and the plate is monitored forradioactivity using a TopCount NXT (Packard Instruments, IL).

Binding experiments are performed in buffer containing 10 mM HEPES, 150mM NaCl, and 0.1% BSA, pH=7.5. Membranes are incubated with 50 μl [¹²⁵I]His⁵, D-Tyr⁶ GnRH (0.2 nM final concentration) and 50 μl of smallmolecule competitors at concentrations ranging from 30 pM to 10 μM for atotal volume in each well of 200 μl. Incubations are carried out for 2hrs at room temperature. The reaction is terminated by rapid filtrationover GF/C filters as previously described. Curve fitting is performedusing Excel Fit Software (IDBS, Emeryville, Calif.). The Ki values arecalculated using the method of Cheng and Prusoff (Cheng and Prusoff,1973) using a Kd value of 0.7 nM for the radioligand which waspreviously determined in saturation binding experiments.

Ca⁺⁺ Flux Measurement

To determine the inhibition of GnRH-stimulated calcium flux in cellsexpressing the human GnRH receptor, a 96-well plate is seeded with RBLcells stably transfected with the human GnRH receptor at a density of50,000 cells/well and allowed to attach overnight. Cells are loaded for1 hr at 37° C. in the following medium: DMEM with 20 mM HEPES, 10% FBS,2 μM Fluo-4, 0.02% pluronic acid and 2.5 mM probenecid. Cells are washed4 times with wash buffer (Hanks balanced salt, 20 mM HEPES, 2.5 mMprobenecid) after loading, leaving 150 μl in the well after the lastwash. GnRH is diluted in 0.1% BSA containing FLIPR buffer (Hanksbalanced salt, 20 mM HEPES) to a concentration of 20 nM and dispensedinto a 96-well plate (Low protein binding). Various concentrations ofantagonists are prepared in 0.1% BSA/FLIPR buffer in a third 96-wellplate. Measurement of fluorescence due to GnRH stimulated (50 μl of 20nM, or 4 nM final) Ca⁺⁺ flux is performed according to manufacturer'sinstructions on a FLIPR system (Molecular Devices, FLIPR384 system,Sunnyvale, Calif.) following a 1-minute incubation with 50 μl ofantagonist at varying concentrations.

Phosphoinositol Hydrolysis Assay

The procedure is modified from published protocols (W. Zhou et al; J.Biol. Chem. 270(32), pp18853-18857, 1995). Briefly, RBL cells stablytransfected with human GnRH receptors are seeded in 24 well plates at adensity of 200,000 cell/well for 24 hrs. Cells are washed once withinositol-free medium containing 10% dialyzed FBS and then labeled with 1uCi/mL of [myo-³H]-inositol. After 20-24 hrs, cells are washed withbuffer (140 nM NaCl, 4 mM KCl, 20 mM Hepes, 8.3 mM glucose, 1 mM MgCl₂,1 mM CaCl₂ and 0.1% BSA) and treated with native GnRH peptide in thesame buffer with or without various concentrations of antagonist and 10mM LiCl for 1 hour at 37° C. Cells are extracted with 10 mM formic acidat 4° C. for 30 min and loaded on a Dowex AG1-X8 column, washed andeluted with 1 M ammonium formate and 0.1 M formic acid. The eluate iscounted in a scintillation counter. Data from PI hydrolysis assay areplotted using non-linear least square regression by the Prism program(Graphpad, GraphPad Software, San Diego, Calif.), from which dose ratiois also calculated. The Schild linear plot is generated from thedose-ratios obtained in four independent experiments by linearregression, and the X-intercept is used to determine the affinity of theantagonist.

Castrate Animal Studies

Studies of castrate animals provide a sensitive in vivo assay for theeffects of GnRH antagonist (Andrology 25: 141-147, 1993). GnRH receptorsin the pituitary gland mediate GnRH-stimulated LH release into thecirculation. Castration results in elevated levels of circulating LH dueto reduction of the negative feedback of gonadal steroids resulting inenhancement of GnRH stimulated LH release. Consequently, measurement ofsuppression of circulating LH levels in castrated macaques can be usedas a sensitive in vivo measure of GnRH antagonism. Therefore, malemacaques are surgically castrated and allowed to recover for four-weeksat which point elevated levels of LH are present. Animals are thenadministered the test compound as an oral or i.v. dose and serial bloodsamples taken for measurement of LH. LH concentrations in serum fromthese animals can be determined by immunoassay or bioassay techniques(Endocrinology 107: 902-907, 1980).

Preparation of GnRH Radioligand

The GnRH analog is labeled by the chloramine-T method. To 10 μg ofpeptide in 20 μl of 0.5M sodium phosphate buffer, pH 7.6, is added 1 mCiof Na¹²⁵I, followed by 22.5 μg chloramine-T in 15 μl 0.05M sodiumphosphate buffer and the mixture is vortexed for 20 sec. The reaction isstopped by the addition of 60 μg sodium metabisulfite in 30 μl 0.05Msodium phosphate buffer and the free iodine is removed by passing thereaction mixture through a C-8 Sep-Pak cartridge (Millipore Corp.,Milford, Mass.). The peptide is eluted with a small volume of 80%acetonitrile/water. The recovered labeled peptide is further purified byreverse phase HPLC on a Vydac C-18 analytical column (The SeparationsGroup, Hesperia, Calif.) on a Beckman 334 gradient HPLC system using agradient of acetonitrile in 0.1% TFA. The purified radioactive peptideis stored in 0.1% BSA/20% acetonitrile/0.1% TFA at −80° C. and can beused for up to 4 weeks.

RIA of LH and FSH

For determination of the LH levels, each sample medium is assayed induplicates and all dilutions are done with RIA buffer (0.01M sodiumphosphate buffer/0.15M NaCl/1% BSA/0.01% NaN3, pH 7.5) and the assay kitis obtained from the Nation Hormone and Pituitary Program supported byNIDDK. To a 12×75 mm polyethylene test tube is added 100 μl of samplemedium diluted 1:5 or rLH standard in RIA buffer and 100 μl of[125I]-labeled rLH (˜30,000 cpm) plus 100 μl of rabbit anti-rLH antibodydiluted 1:187,500 and 100 μl RIA buffer. The mixture is incubated atroom temperature over-night. In the next day, 100 μl of goat anti-rabbitIgG diluted 1:20 and 100 μl of normal rabbit serum diluted 1:1000 areadded and the mixture incubated for another 3 hr at room temperature.The incubated tubes are then centrifuged at 3,000 rpm for 30 min and thesupernatant removed by suction. The remaining pellet in the tubes iscounted in a gamma-counter. RIA of FSH is done in a similar fashion asthe assay for LH with substitution of the LH antibody by the FSHantibody diluted 1:30,000 and the labeled rLH by the labeled rFSH.

Activity of GnRH Receptor Antagonists

Activity of GnRH receptor antagonists are typically calculated from theIC₅₀ as the concentration of a compound necessary to displace 50% of theradiolabeled ligand from the GnRH receptor, and is reported as a “K_(i)”value calculated by the following equation:$K_{i} = \frac{{IC}_{50}}{1 + {L/K_{D}}}$where L=radioligand and K_(D)=affinity of radioligand for receptor(Cheng and Prusoff, Biochem. Pharmacol. 22:3099, 1973). GnRH receptorantagonists of this invention have a K_(i) of 100 μM or less. In apreferred embodiment of this invention, the GnRH receptor antagonistshave a K_(i) of less than 10 μM, and more preferably less than 1 μM, andeven more preferably less than 0.1 μM (i.e., 100 nM). To this end, allcompounds specifically disclosed in the Examples have K_(i)'s of lessthan 100 nM in one or more of Membrane Binding Assays 1 through 3 above.

The ability of the GnRH antagonists to inhibit the major drugmetabolizing enzymes in the human liver, namely, CYP2D6 and CYP3A4, canbe evaluated in vitro according to a microtiter plate-based fluorimetricmethod described by Crespi et al. (Anal. Biochem. 248: 188-190; 1997).AMMC (i.e.,3-[2-(N,N-Diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin)and BFC (i.e., 7-benzyloxy-4-(trifluoromethyl)coumarin) at aconcentration equal to Km (that is, the concentration of substrate thatproduces one half of the maximal velocity) are used as marker substratesfor CYP2D6 and CYP3A4, respectively. Briefly, recombinant CYP2D6 orCYP3A4 is incubated with marker substrate and NADPH generating system(consisting of 1 mM NADP+, 46 mM glucose-6-phosphate and 3 units/mLglucose-6-phosphate dehydrogenase) at 37° C., in the absence or presenceof 0.03, 0.09, 0.27, 0.82, 2.5, 7.4, 22, 67 and 200 μM of a sample GnRHantagonist. Reactions are stopped by the addition of an equal volume ofacetonitrile. The precipitated protein is removed by centrifugation andthe clear supernatant fluid is analyzed using a microtiter platefluorimeter. GnRH antagonists of the present invention preferably haveK_(i)'s greater than 250 nM, more preferably greater than 1 μM and mostpreferably greater than 5 μM.

As mentioned above, the GnRH receptor antagonists of this invention haveutility over a wide range of therapeutic applications, and may be usedto treat a variety of sex-hormone related conditions in both men andwomen, as well as mammals in general. For example, such conditionsinclude endometriosis, uterine fibroids, polycystic ovarian disease,hirsutism, precocious puberty, gonadal steroid-dependent neoplasia suchas cancers of the prostate, breast and ovary, gonadotrophe pituitaryadenomas, sleep apnea, irritable bowel syndrome, premenstrual syndrome,benign prostatic hypertrophy, contraception and infertility (e.g.,assisted reproductive therapy such as in vitro fertilization).

The compounds of this invention are also useful as an adjunct totreatment of growth hormone deficiency and short stature, and for thetreatment of systemic lupus erythematosis.

In addition, the compounds are useful in combination with androgens,estrogens, progesterones, and antiestrogens and antiprogestogens for thetreatment of endometriosis, fibroids, and in contraception, as well asin combination with an angiotensin-converting enzyme inhibitor, anangiotensin II-receptor antagonist, or a renin inhibitor for thetreatment of uterine fibroids. The compounds may also be used incombination with bisphosphonates and other agents for the treatmentand/or prevention of disturbances of calcium, phosphate and bonemetabolism, and in combination with estrogens, progesterones and/orandrogens for the prevention or treatment of bone loss or hypogonadalsymptoms such as hot flashes during therapy with a GnRH antagonist.

In another embodiment of the invention, pharmaceutical compositionscontaining one or more GnRH receptor antagonists are disclosed. For thepurposes of administration, the compounds of the present invention maybe formulated as pharmaceutical compositions. Pharmaceuticalcompositions of the present invention comprise a GnRH receptorantagonist of the present invention and a pharmaceutically acceptablecarrier and/or diluent. The GnRH receptor antagonist is present in thecomposition in an amount which is effective to treat a particulardisorder—that is, in an amount sufficient to achieve GnRH receptorantagonist activity, and preferably with acceptable toxicity to thepatient. Typically, the pharmaceutical compositions of the presentinvention may include a GnRH receptor antagonist in an amount from 0.1mg to 250 mg per dosage depending upon the route of administration, andmore typically from 1 mg to 60 mg. Appropriate concentrations anddosages can be readily determined by one skilled in the art.

Pharmaceutically acceptable carrier and/or diluents are familiar tothose skilled in the art. For compositions formulated as liquidsolutions, acceptable carriers and/or diluents include saline andsterile water, and may optionally include antioxidants, buffers,bacteriostats and other common additives. The compositions can also beformulated as pills, capsules, granules, or tablets which contain, inaddition to a GnRH receptor antagonist, diluents, dispersing and surfaceactive agents, binders, and lubricants. One skilled in this art mayfurther formulate the GnRH receptor antagonist in an appropriate manner,and in accordance with accepted practices, such as those disclosed inRemington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co.,Easton, Pa. 1990.

In another embodiment, the present invention provides a method fortreating sex-hormone related conditions as discussed above. Such methodsinclude administering of a compound of the present invention to awarm-blooded animal in an amount sufficient to treat the condition. Inthis context, “treat” includes prophylactic administration. Such methodsinclude systemic administration of a GnRH receptor antagonist of thisinvention, preferably in the form of a pharmaceutical composition asdiscussed above. As used herein, systemic administration includes oraland parenteral methods of administration. For oral administration,suitable pharmaceutical compositions of GnRH receptor antagonistsinclude powders, granules, pills, tablets, and capsules as well asliquids, syrups, suspensions, and emulsions. These compositions may alsoinclude flavorants, preservatives, suspending, thickening andemulsifying agents, and other pharmaceutically acceptable additives. Forparental administration, the compounds of the present invention can beprepared in aqueous injection solutions which may contain, in additionto the GnRH receptor antagonist, buffers, antioxidants, bacteriostats,and other additives commonly employed in such solutions.

The following example is provided for purposes of illustration, notlimitation. In summary, the GnRH receptor antagonists of this inventionmay be assayed by the general methods disclosed above, while thefollowing Examples disclose the synthesis of representative compounds ofthis invention.

EXAMPLES

A. HPLC Methods for Analyzing the Samples

-   -   Retention time, t_(R), in minutes        Method 1—Supercritical Fluid Chromatography Mass Spectrum        (SFC-MS)

-   Column: 4.6×150 mm Deltabond Cyano 5 μM from    Thermo-Hypersil-Keystone.

-   Mobile phase: SFC grade carbon dioxide and optima grade methanol    with 1 mM disodium diethylmalonate modifier.

-   Temperature: 50° C.

-   Pressure: 120 bar

-   Flow Rate: 4.8 mL/min

-   Gradient: 5% to 55% methanol over 1.7 min and hold at 55% for 0.8    min then return to 5% in 0.1 min for total run time of 2.6 min    Method 2 (HPLC-MS)

-   Column: Waters ODS-AQ, 2.0×50 mm

-   Mobile phase: A=water with 0.05% trifluoroacetic acid;    B=acetonitrile with 0.05% trifluoroacetic acid

-   Gradient: 95% A/5% B to 5% A/95% B over 13.25 min and hold 5% A/95%    B over 2 min then return to 95% A/5% B over 0.25 min.

-   Flow Rate: 1 mL/min

-   UV wavelength: 220 and 254 nM    Method 3 (HPLC-MS)

-   Column: BHK Lab ODS-O/B, 4.6×50 mm, 5 μM

-   Mobile phase: A=water with 0.05% trifluoroacetic acid;    B=acetonitrile with 0.05% trifluoroacetic acid

-   Gradient: 95% A/5% B for 0.5 min, then to 90% A/10% B for 0.05 min.    from 90% A/10% B to 5% A/95% B over 18.94 min, then to 1% A/99% B    over 0.05 min and hold 1% A/99% B over 2.16 min. then return to    95%/5% B over 0.50 min.

-   Flow rate: 2.5 mL/min.

-   UV wavelength: 220 and 254 nM    Method 4 (HPLC-MS)

-   Column: Waters ODS-AQ, 2.0×50 mm

-   Mobile phase: A=water with 0.05% trifluoroacetic acid;    B=acetonitrile with 0.05% trifluoroacetic acid

-   Gradient: 95% A/5% B to 10% A/90% B over 2.25 min and hold 10% A/90%    B over 1.0 min then return to 95% A/5% B over 0.1 min.

-   Flow Rate: 1 mL/min

-   UV wavelength: 220 and 254 nM    Method 5 (HPLC)

-   Column: Agilent, Zorbax SB-C18, 5 μM, 4.6×250 mm.

-   Mobile phase: A=water with 0.05% trifluoroacetic acid;    B=acetonitrile with 0.05% trifluoroacetic acid

-   Gradient: 95% A/5% B to 5% A/95% B over 50 min, then 5% A/95% B to    1% A/99% B over 0.1 min, then hold 1% A/99% for 0.8 min and back to    95% A/5% over 0.2 min, hold such gradient for 4 min.

-   Flow rate 2.0 mL/min.

-   UV wavelength: 220 and 254 nM    Method 6 (HPLC-MS)

-   Column: Phenomenex Synergi 4μ Max-RP 80A, 50.0×2.0 mm

-   Mobile Phase: A=water with 0.025% of trifluoroacetic acid;    B=acetonitrile with 0.025% of trifluoroacetic acid

-   Gradient: 95% A/5% B 0.25 min, then 95% A/5% B to 95% B/5% A over 13    min, maintaining 95% A/5% B to 95% B/5% A over 2 min, then back to    95% A/5% B in 0.25 min.

-   Flow rate: 1 mL/min

-   UV wavelength: 220 nM and 254 nM

Example 13-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione

Step 1A: Preparation of 2-fluoro-6-(trifluoromethyl)benzylamine 1a

To 2-fluoro-6-(trifluoromethyl)benzonitrile (45 g, 0.238 mmol) in 60 mLof THF was added 1 M BH₃:THF slowly at 60° C. and the resulting solutionwas refluxed overnight. The reaction mixture was cooled to ambienttemperature. Methanol (420 mL) was added slowly and stirred well. Thesolvents were then evaporated and the residue was partitioned betweenEtOAc and water. The organic layer was dried over Na₂SO₄. Evaporationgave 1a as a yellow oil (46 g, 0.238 mmol). MS (CI) m/z 194.0 (MH⁺).

Step 1B: Preparation of N-[2-fluoro-6-(trifluoromethyl)benzyl]urea 1b

To 2-fluoro-6-(trifluoromethyl)benzylamine 1a (51.5 g, 0.267 mmol) in aflask, urea (64 g, 1.07 mmol), HCl (conc., 30.9 mmol, 0.374 mmol) andwater (111 mL) were added. The mixture was refluxed for 6 hours. Themixture was cooled to ambient temperature, further cooled with ice andfiltered to give a yellow solid. Recrystallization with 400 mL of EtOAcgave 1b as a white solid (46.2 g, 0.196 mmol). MS (CI) m/z 237.0 (MH⁺).

Step 1C: Preparation of1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methylpyrimidine-2,4(1H,3H)-dione1c

NaI (43.9 g, 293 mmol) was added toN-[2-fluoro-6-(trifluoromethyl)benzyl]urea 1b (46.2 g, 19.6 mmol) in 365mL of acetonitrile. The resulting mixture was cooled in an ice-waterbath. Diketene (22.5 mL, 293 mmol) was added slowly via dropping funnelfollowed by addition of TMSCl (37.2 mL, 293 mmol) in the same manner.The resulting yellow suspension was allowed to warm to room temperatureslowly and was stirred for 20 hours. LC-MS showed the disappearance ofstarting material. To the yellow mixture 525 mL of water was added andstirred overnight. After another 20 hours stirring, the precipitate wasfiltered via Buchnner funnel and the yellow solid was washed with waterand EtOAc to give 1c as a white solid (48.5 g, 16 mmol). ¹H NMR (CDCl₃)δ 2.15 (s, 3H), 5.37 (s, 2H), 5.60 (s, 1H), 7.23-7.56 (m, 3H), 9.02 (s,1H); MS (CI) m/z 303.0 (MH⁺).

Step 1D: Preparation of5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methylpyrimidine-2,4(1H,3H)-dione1d

Bromine (16.5 mL, 0.32 mmol) was added to1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methylpyrimidine-2,4(1H,3H)-dione1c (48.5 g, 0.16 mol) in 145 mL of acetic acid. The resulting mixturebecame clear then formed precipitate within an hour. After 2 hoursstirring, the yellow solid was filtered and washed with cold EtOAc to analmost white solid. The filtrate was washed with sat. NaHCO₃ and driedover Na₂SO₄. Evaporation gave a yellow solid which was washed with EtOACto give a light yellow solid. The two solids were combined to give 59.4g of 1d (0.156 mol) total. ¹H NMR (CDCl₃) δ 2.4 (s, 3H), 5.48 (s, 2H),7.25-7.58 (m, 3H), 8.61 (s, 1H); MS (CI) m/z 380.9 (MH⁺).

5-Bromo-1-[2,6-difluorobenzyl]-6-methylpyrimidine-2,4(1H,3H)-dione 1d.1was made using the same procedure.

Step 1E: Preparation of5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-3-[2(R)-tert-butoxycarbonylamino-2-phenylethyl]-pyrimidine-2,4(1H,3H)-dione1e

To5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methylpyrimidine-2,4(1H,3H)-dione1d (15 g, 39.4 mmol) in 225 mL of THF were addedN-t-Boc-D-phenylglycinol (11.7 g, 49.2 mmol) and triphenylphosphine(15.5 g, 59.1 mmol), followed by addition of di-tert-butylazodicarboxylate (13.6 g, 59.1 mmol). The resulting yellow solution wasstirred overnight. The volatiles were evaporated and the residue waspurified by silica gel with 3:7 EtOAc/Hexane to give 1e as a white solid(23.6 g, 39.4 mmol). MS (CI) m/z 500.0 (MH⁺-Boc).

Step 1F: Preparation of3-[2(R)-amino-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione1f

To5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-3-[2(R)-tert-butoxycarbonylamino-2-phenylethyl]-pyrimidine-2,4(1H,3H)-dione1e (15 g, 25 mmol) in 30 mL/90 mL of H₂O/dioxane in a pressure tube wereadded 2-fluoro-3-methoxyphenylboronic acid (4.25 g, 25 mmol) and sodiumcarbonate (15.75 g, 150 mmol). N₂ gas was bubbled through for 10 min.Tetrakis(triphenylphosphine)palladium (2.9 g, 2.5 mmol) was added, thetube was sealed and the resulting mixture was heated with stirring at90° C. overnight. After cooling to ambient temperature, the precipitatewas removed by filtration. The volatiles were removed by evaporation andthe residue was partitioned between EtOAc/sat. NaHCO₃. The organicsolvent was evaporated and the residue was chromatographed with 2:3EtOAc/Hexane to give 13.4 g (20.8 mmol, 83%) yellow solid.

This yellow solid (6.9 g, 10.7 mmol) was dissolved in 20 mL/20 mLCH₂Cl₂/TFA. The resulting yellow solution was stirred at roomtemperature for 2 hours. The volatiles were evaporated and the residuewas partitioned between EtOAc/sat. NaHCO₃. The organic phase was driedover Na₂SO₄. Evaporation gave 1f as a yellow oil (4.3 g, 7.9 mmol, 74%).¹H NMR (CDCl₃) δ 2.03 (s, 3H), 3.72-4.59 (m, 6H), 5.32-5.61 (m, 2H),6.74-7.56 (m, 11H); MS (CI) m/z 546.0 (MH⁺).

3-[2(R)-amino-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione1f.1 was made using the same procedure described in this example.

Step 1G: Preparation of3-[2(R)-{ethoxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione1g

To compound3-[2(R)-amino-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione1 f (5 g, 9.4 mmol) in 100 mL of acetonitrile were added ethyl4-bromobutyrate (4 mL, 28.2 mmol) and Hunig's base (1.6 mL, 9.4 mmol).After reflux at 95° C. overnight, the reaction mixture was cooled toambient temperature and the volatiles were removed. The residue waschromatographed with 10:10:1 EtOAc/Hexane/Et₃N to give 1g as a yellowoil (3.0 g, 4.65 mmol). MS (CI) m/z 646.2 (MH⁺).

Step 1H: Preparation of3-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione1-1

Compound3-[2(R)-{ethoxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione1g (2.6 g, 4.0 mmol) was dissolved in 30 mL/30 mL of THF/water. SolidNaOH (1.6 g, 40 mmol) was added and the resulting mixture was heated at50° C. overnight. The mixture was cooled to ambient temperature and thevolatiles were evaporated. Citric acid was added to the aqueous solutionuntil pH=3. Extraction with EtOAc followed by evaporation of solventgave 1.96 g of a white gel. The gel was passed through a Dowex MSC-1macroporous strong cation-exchange column to convert to sodium salt.Lyopholization gave white solid 1-1 as the sodium salt (1.58 g, 2.47mmol). ¹H NMR (CD₃OD) δ 1.69-1.77 (m, 2H), 2.09 (s, 3H), 2.09-2.19 (t,J=7.35 Hz, 2H), 2.49-2.53 (t, J=7.35H, 2H), 3.88 (s, 3H), 4.15-4.32 (m,3H), 5.36-5.52 (m, 2H), 6.60-7.63 (m, 11H); HPLC-MS (CI) m/z 632.2(MH⁺), t_(R)=26.45, (method 5)

The following compounds were synthesized according to the aboveprocedure.

t_(R) No. —N(R₅)—X—R₆ M.W. Mass (Method #) 1-1

631.60 632.2 26.45 (5) 1-2

617.57 618.0 2.777 (4) 1-3

645.62 646.0 2.789 (4)

Step 1I: Preparation of3-[2(R)-[N-methyl-N-hydroxycarbonylpropyl-amino]-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione1-4

To compound 1-1 (0.045 mmol) in 1 mL MeOH, formaldehyde (0.0475 mmol)was added followed by addition of 8 M BH₃:Pyridine (0.0475 mmol). Afterovernight shaking, compound 1-4 was purified by prep. LC-MS. HPLC-MS(CI) m/z 646.5 (MH⁺), t_(R)=2.231, (method 4)

The following compounds were synthesized according to the aboveprocedure.

t_(R) No. —N(R₅)—X—R₆ R₄ M.W. Mass (Method #) 1-4

Ph 645.62 646.2 2.231 (4) 1-5

Ph 659.65 660.2 2.235 (4) 1-6

cyclopentyl 637.64 638.3 2.259 (4) 1-7

cyclopentyl 651.67 652.3 2.294 (4) 1-8

isobutyl 625.63 626.0 2.594 (4)

Example 23-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione

Step 2A: Preparation of5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-3-[2(R)-amino-2-phenylethyl]-pyrimidine-2,4(1H,3H)-dione2a

5-Bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-3-[2(R)-tert-butoxycarbonylamino-2-phenylethyl]-pyrimidine-2,4(1H,3H)-dione1e was dissolved in 20 mL/20 mL CH₂Cl₂/TFA. The resulting yellowsolution was stirred at room temperature for 2 hours. The volatiles wereevaporated and the residue was partitioned between EtOAc/sat. NaHCO₃.The organic phase was dried over Na₂SO₄. Evaporation gave 2a as a yellowoil.

Step 2B: Preparation of5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-3-[2(R)-amino-2-phenylethyl]-pyrimidine-2,4(1H,3H)-dione2b

To compound 2a (40 mg, 0.08 mmol) in 0.25 mL/0.75 mL of H₂O/dioxane in a4 mL vial was added 2-chlorophenyl boronic acid (0.12 mmol) and sodiumcarbonate (51 mg, 0.48 mmol, 6 eq). Nitrogen gas was bubbled through thesolution for 1 minute and tetrakis(triphenylphosphine)palladium (9.24mg, 0.008 mmol) was added. The resulting mixture was sealed and heatedat 90° C. overnight. After cooling to ambient temperature, theprecipitate was removed by filtration and was purified by prep. LC-MS togive 2b.

Step 2C:3-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione2-1

To compound 2b (0.03 mmol) in 1 mL MeOH, succinic semialdehyde (0.03mmol) was added followed by addition of 8 M BH₃:Pyridine (0.03 mmol).After overnight shaking, the compound 2-1 was purified by prep. LC-MS.MS (CI) m/z 618.2 (MH⁺) t_(R)=1.005 (method 1)

The following compounds were synthesized according to the aboveprocedure.

t_(R) No. R_(2a) R_(1a) R_(1b) M.W. Mass (Method #) 2-1 CF₃ 2-Cl H618.02 618.2 1.005 (1) 2-2 CF₃ 2-F H 601.57 602.2 0.976 (1) 5.194 (6)2-3 CF₃ H H 583.58 584.3 1.000 (1) 5.572 (6) 2-4 CF₃ 3-isopropyl H625.66 626.3 6.882 (1) 2-5 CF₃ 3-ethoxy H 627.63 628.3 0.913 (1) 2-6 CF₃3,4-methyl-enedioxy 627.59 628.2 0.932 (1) 2-7 CF₃ 2-F 3-OH 617.57 618.20.979 (1) 2-8 CF₃ 3-methyl H 597.61 598.2 5.455 (6) 2-9 SO₂CH₃ 2-F3-methoxy 641.69 642.1 4.820 (6) 2-10 F 2-F 3-methoxy 581.59 582.2 5.532(6) 2-11 CF₃ 3-Cl H 618.02 617.9 5.216 (6) 2-12 CF₃ 3,4-O—CH₂—CH₂—625.62 626.0 4.774 (6) 2-13 CF₃ 2-F 3-methyl 615.60 616.2 6.381 (6) 2-14CF₃ 3-isopropyloxy H 641.66 642.2 6.676 (6)

Example 33-[2(R)-{2-[5-tetrazoylpropyl]-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione

Step 3A:3-[2(R)-{3-cyanopropyl-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione3a

Compound 1f (110 mg, 0.2 mmol) was dissolved in acetonitrile (5 mL) anddiisopropylethyl amine (52 mg, 0.4 mmol) was added, followed by theaddition of 4-bromobutyronitrile (90 mg, 0.6 mmol). The reaction mixturewas refluxed for 16 hours. Volatiles were evaporated and the residue waspurified by flash chromatography (silica, 5% MeOH/CH₂Cl₂) to givecompound 3a (115 mg, 94%). MS (CI) m/z 613.3 (MH⁺).

Step 3B:3-[2(R)-{2-[5-tetrazoylpropyl]-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione3-1

A solution of 3a (38 g, 0.06 mmol) in toluene (5 mL) was addedtributyltin azide (42 mg, 0.12 mmol), and the reaction mixture washeated at 100° C. for 14 hours. The mixture was cooled, partitionedbetween EtOAc and 1 N NaOH, and the organic layer was washed with 1 NHCl and brine. The organic layer was dried (sodium sulfate), evaporated,and the residue was purified by flash chromatography (silica, 7%MeOH/CH₂Cl₂) to give compound 3-1 (10 mg, 25%). HPLC-MS (CI) m/z 656.2(MH⁺), t_(R)=2.128 min, (method 4)

Example 43-[2(R)-{hydroxycarbonylpropyl-amino}-2-cyclohexylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione

Step 4A: Preparation of tert-butyl 1-cyclohexyl-2-hydroxyethylcarbamate4a

A solution of N-(t-butyloxycarbonyl)cyclohexylglycine (2.0 g, 7.77 mmol)in anhydrous THF (10 mL) was cooled to 0° C. Borane solution (1 M inTHF, 15.5 mL, 15.5 mmol) was added slowly and the reaction mixture waswarmed to room temperature and stirred for 2 hours. The reaction wasquenched with MeOH (5 mL), volatiles were evaporated and the residue waspartitioned between water and EtOAc. The organic layer was washed withsaturated NaHCO₃/water, brine, dried (sodium sulfate), and evaporated togive tert-butyl 1-cyclohexyl-2-hydroxyethylcarbamate 4a (1.26 g, 66.7%),MS (CI) m/z 144.2 (MH⁺-Boc).

Step 4B: Preparation of5-bromo-3-[2(R)-tert-butoxycarbonylamino-2-cyclohexylethyl]-1-[2,6-difluorobenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione4b

A solution of tert-butyl 1-cyclohexyl-2-hydroxyethylcarbamate 4a (638mg, 2.62 mmol) in THF (10 mL) was treated with5-bromo-1-(2,6-difluorobenzyl)-6-methylpyrimidine-2,4(1H,3H)-dione 1d.1(869 mg, 2.62 mmol) and triphenylphosphine (1.03 g, 3.93 mmol) atambient temperature, then di-tert-butylazodicarboxylate (906 mg, 3.93mmol) was introduced. The reaction mixture was stirred at ambienttemperature for 16 hours and volatiles were evaporated. The residue waspartitioned between saturated NaHCO₃/H₂O and EtOAc. The organic layerwas dried (sodium sulfate), evaporated, and purified by flashchromatography (silica, 25% EtOAc/hexanes) to give compound 4b (1.39 g,95.4%). MS (CI) m/z 456.1, 458.1 (MH⁺-Boc).

Step 4C: Preparation of3-[2(R)-tert-butoxycarbonylamino-2-cyclohexylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione4c

5-Bromo-3-[2(R)-tert-butoxycarbonylamino-2-cyclohexylethyl]-1-[2,6-difluorobenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione4b (1.0 g, 1.79 mmol) in benzene/EtOH/ethylene glycol dimethyl ether(20/2/22 mL) was added 2-fluoro-3-methoxyphenylboronic acid (382 mg,2.24 mmol) and saturated Ba(OH)₂/water (˜0.5 M, 15 mL). The reactionmixture was deoxygenated with N₂ for 10 minutes,tetrakis(triphenylphosphine)palladium (0) (208 mg, 0.18 mmol) was addedand the reaction mixture was heated at 80° C. overnight under N₂. Thereaction mixture was partitioned between brine and EtOAc. The organiclayer was dried (sodium sulfate), evaporated, and purified by flashchromatography (silica, 30% EtOAc/hexanes) to give compound 4c (348 mg,32.3%). MS (CI) m/z 502.2 (MH⁺-Boc).

Step 4D: Preparation of3-[2(R)-amino-2-cyclohexylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione4d

To compound 4c (300 mg, 0.5 mmol) in dichloromethane (2 mL) was addedTFA (2 mL) and the reaction mixture was stirred at ambient temperaturefor 1 hour. Volatiles were evaporated and the residue was partitionedbetween saturated NaHCO₃/water and EtOAc. The organic layer was dried(sodium sulfate), evaporated, purified by reverse phase HPLC (C-18column, 15-75% ACN/water) to give compound 4d. MS (CI) m/z 502.2 (MH⁺).

Step 4E: Preparation of3-[2(R)-{hydroxycarbonylpropyl-amino}-2-cyclohexylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-{[2,6-difluorobenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione4-1

A solution of compound 4d (10 mg, 0.02 mmol) in methanol (2 mL) wasadded succinic semialdehyde (15 mg, 15% aqueous solution), followed bythe addition of borane/pyridine (8 M, 3 μL). The reaction mixture wasstirred at ambient temperature for 1 hour. Volatiles were evaporated andthe residue was purified directly on preparative TLC plate eluting with7% MeOH/CH₂Cl₂ to give compound 4-1 (5 mg). MS (CI) m/z 588.3 (MH⁺).

3-[2(R)-{hydroxycarbonylpropyl-amino}-2-cyclohexylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione4-2 was synthesized using the same procedure and intermediate 1d.

The following compounds were synthesized according to the aboveprocedure.

t_(R) No. —N(R₅)—X—R₆ R_(2a) R₄ MW Mass (Method #) 4-1

F cyclohexyl 587.63 588.4 5.350 (3) 4-2

CF₃ cyclohexyl 637.64 638.3 27.56 (5) 4-3

CF₃ cyclopentyl 623.60 624.2 2.290 (4) 4-4

CF₃ isobutyl 611.61 612.3 6.480 (6) 4-5

CF₃ cyclohexyl 651.67 652.1 2.340 (4) 4-6

CF₃ isobutyl 625.63 626.0 2.593 (4) 4-7

CF₃ isobutyl 639.66 640.0 2.61 (4) 4-8

CF₃ isobutyl 597.58 598.0 2.571 (4)

Example 53-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione

Step 5A: Preparation of5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione5a

A suspension of 5-bromouracil (31.0 g) in 300 mL of dichloroethane istreated with N,O-bis(trimethylsilyl)acetamide (80 mL). The reactionmixture is heated under nitrogen. The solution is cooled to ambienttemperature, 2-fluoro-6-(trifluoromethyl)benzyl bromide (50 g) is addedand the reaction mixture is heated overnight under the nitrogen. Thereaction is cooled, quenched with MeOH, and partitioned betweendichloromethane and water. The organic layer is washed with brine, dried(sodium sulfate), and evaporated to give a solid. The crude product istriturated with ether, filtered, and washed with ether three timesproviding 40.7 g of5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione5a. MS (CI) m/z 366.0, 368.0 (MH⁺).

Step 5B: Preparation of3-[2(R)-amino-2-phenylethyl]-5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione5b

A solution of5-bromo-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione5a (19.2 g, 52.3 mmol) in THF (180 mL) was treated withN-(t-butyloxycarbonyl)-D-α-phenylglycinol (13.6 g, 57.5 mmol) andtriphenylphosphine (20.6 g, 78.5 mmol) at room temperature, thendi-tert-butylazodicarboxylate (18.0 g, 78.5 mmol) was introduced inseveral portions over 5 minutes. The mixture was stirred at roomtemperature for 16 hour, additional THF (90 mL) was added, and themixture was heated to 50° C. Concentrated HCl (34.6 mL, 418 mmol) wasadded, and the reaction mixture was stirred at 50° C. for 40 hours.After dilution with ethyl acetate (100 mL), the solid was filtered,washed with additional ethyl acetate (100 mL), and dried to givecompound 5b (26.9 g, 98%) as a white powder. MS (CI) m/z 485.0, 487.0(MH⁺).

Step 5C: Preparation of3-[2(R)-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione5c

To compound 5b (10.45 g, 20 mmol) in dioxane/water (180/20 mL) was added2-chlorophenylboronic acid (6.26 g, 40 mmol) and Na₂CO₃ (12.72 g, 120mmol). The mixture was deoxygenated with N₂ for 15 minutes,tetrakis(triphenylphosphine) palladium (0) (2.31 g, 2 mmol) was addedand the reaction mixture was heated at 90° C. for 16 hours. The reactionwas partitioned between EtOAc and H₂O. The organic layer was washed withbrine, dried over Na₂SO₄, concentrated and purified by columnchromatography on silica gel with ethyl acetate/hexanes/triethylamine500/500/6 to 800/200/7 to afford compound 5c (7.26 g, 70%) as a whitefoam. MS (CI) m/z 518.0, 520.1 (MH⁺).

Step 5D: Preparation of3-[2(R)-{ethoxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione5d

A mixture of compound 5c (4.1 g, 7.93 mmol), ethyl 4-bromobutyrate (3.6mL, 23.79 mmol) and K₂CO₃ (2.2 g, 15.86 mmol) in MeCN (80 mL) wasrefluxed for 16 hours. MeCN was removed, and the residue was partitionedbetween EtOAc and H₂O. The organic layer was washed with brine, driedover Na₂SO₄, concentrated and purified by column chromatography onsilica gel with ethyl acetate/hexanes/triethylamine 400/600/7 to affordcompound 5d (2.5 g, 50%) as a yellowish syrup. MS (CI) m/z 632.2, 634.2(MH⁺).

Step 5E: Preparation of3-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione5-1

To compound 5d (2.4 g, 3.8 mmol) was added THF (30 mL) and H₂O (30 mL)followed by NaOH (1.588 g, 39.7 mmol). The mixture was stirred at 50° C.for 16 hours. THF was removed in vacuo, the aq. solution was washed withether, and cooled at 0° C. Neutralization with 10% aq. citric acid (26.0mL, 40.6 mmol) gave a precipitate, which was washed with H₂O and driedto give compound 5-1 (1.88 g, 82%). HPLC-MS (CI) m/z 604.1, 606.1 (MH⁺),t_(R)=2.511 (method 4), t_(R)=26.98 (method 5)

The following compounds were synthesized according to the aboveprocedure.

t_(R) No. R_(1a) R_(1b) R_(2a) MW Mass (Method #) 5-1 Cl H CF₃ 604.00604.1, 2.511 (4) 606.1 26.98 (5) 5-2 F OCH₃ CF₃ 617.57 618.2 2.482 (4)25.45 (5) 5-3 cyano H CF₃ 594.56 594.9 5.548 (6) 5-4 F CH₃ CF₃ 601.57602.2 6.144 (6) 5-5 Cl CH₃ CF₃ 618.02 617.9 5.104 (6) 5-6 F H CF₃ 587.54588.2 5.172 (6) 5-7 F OCH₃ F 567.56 568.2 2.108 (4) 5-8 Cl H F 553.99554.1 2.137 (4) 5-9 Cl H SO₂CH₃ 614.09 614.2 5.020 (6) 5-10 F OCH₃SO₂CH₃ 627.66 628.2 1.178 (1)

Example 63-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione

Step 6A: Preparation of compound methyl(2-chlorophenyl)acetate 6a

To 2-chlorophenylacetic acid (1.04 g, 6 mmol) in MeOH (25 mL) was addedsulfuric acid (6 drops) and the solution was refluxed for 16 hours.After concentration, the residue was taken up in ethyl acetate andwashed with sat'd aq. NaHCO₃, H₂O and brine. The organic layer was driedover Na₂SO₄ and concentrated to give methyl(2-chlorophenyl)acetate 6a(1.08 g, 97.5%) as a yellowish oil. GCMS (EI) m/z 184, 186 (M⁺).Step 6B: Preparation of methyl2-(2-chlorophenyl)-3-(dimethylamino)acrylate 6b

A solution of methyl(2-chlorophenyl)acetate 6a (1.08 g, 5.85 mmol) inDMFDMA (10 mL, 70.8 mmol) was refluxed for 16 hours. After evaporation,the residue was purified by column chromatography on silica gel withethyl acetate/hexanes 1/3 to 1/2 to afford unreactedmethyl(2-chlorophenyl)acetate 6a (0.67 g, 62%) first, and then methyl2-(2-chlorophenyl)-3-(dimethylamino)acrylate 6b (0.38 g, 27%; 71% basedon recovered starting material) as a colorless syrup. MS (CI) m/z 240.2,242.2 (MH⁺).

Step 6C: Preparation of 5-(2-chlorophenyl)pyrimidine-2,4(1H,3H)-dione 6c

To a mixture of methyl 2-(2-chlorophenyl)-3-(dimethylamino)acrylate 6b(0.26 g, 1.08 mmol), urea (0.2 g, 3.26 mmol) and NaI (0.49 g, 3.26 mmol)in acetonitrile (5 mL) was added TMSCl (0.41 mL, 3.26 mmol). Theresulting mixture was refluxed for 16 hours, cooled to room temperature,and 1.0 M NaOH (8 mL) was added. The resultant solution was stirred for20 hr, and acetonitrile was removed in vacuo. The aq. solution waswashed with ether, cooled in ice bath, and neutralized with 1 N HCl (8mL). The precipitate was filtered, washed with additional H₂O, and driedto give 5-(2-chlorophenyl)pyrimidine-2,4(1H,3H)-dione 6c (0.16 g, 66%)as a white solid. MS (CI) m/z 222.9, 224.9 (MH⁺).

Step 6D: Preparation of5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione6d

To a suspension of 5-(2-chlorophenyl)pyrimidine-2,4(1H,3H)-dione 6c(0.16 g, 0.72 mmol) in acetonitrile (5 mL) was addedbis(trimethylsilyl)acetamide (0.36 mL, 1.44 mmol), and the resultingsolution was refluxed for 1.5 hours. After cooling to room temperature,2-fluoro-3-trifluoromethylbenzyl bromide (0.22 g, 0.86 mmol) was added,and reflux was resumed for 16 hours. The reaction was quenched byaddition of MeOH (5 mL) and stirring for 2 hours. After concentration,the residue was purified by column chromatography on silica gel withethyl acetate/hexanes 1/1 to afford5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione6d (0.25 g, 87%) as a white solid. MS (CI) m/z 398.9, 400.9 (MH⁺).

Step 6E: Preparation of3-[2(R)-{tert-butoxycarbonyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione6e

A mixture of5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione6d (125 mg, 0.32 mmol), K₂CO₃ (130 mg, 0.96 mmol) andN-(t-butyloxycarbonyl)-D-α-phenylglycinol mesylate (0.2 g, 0.64 mmol) inDMF (3 mL) was heated at 75° C. for 16 hours. The reaction was dilutedwith ethyl acetate, washed with H₂O and brine, dried over Na₂SO₄ andconcentrated. The residue was purified by column chromatography onsilica gel with ethyl acetate/hexanes 2/3 to afford compound 6e (144 mg,74%). MS (CI) m/z 518.0, 520.0 (MH⁺-Boc).

Step 6F: Preparation of3-[2(R)-amino-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione6f

To a solution of compound 6e (0.144 g, 0.23 mmol) in DCM (1 mL) wasadded TFA (0.5 mL, 6.5 mmol) and the mixture was stirred at roomtemperature for 1.5 hours. After concentration, the residue was taken upin DCM and sat'd aq. NaHCO₃ was added. The aqueous layer was extractedwith DCM. Combined organic extracts were dried over Na₂SO₄ andconcentrated to give compound 6f (0.12 g). MS (CI) m/z 518.0, 520.1(MH⁺).

Step 6G: Preparation of3-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione6-1

A solution of compound 6f (0.1 g, 0.19 mmol) and succinic semialdehyde(15 wt % solution in water; 0.13 mL, 0.21 mmol) in MeCN was stirred atroom temperature for 5 minutes. Borane pyridine complex (8 M; 72 μL) wasadded and stirred for 16 hours. After concentration, the residue waspurified first on prep TLC plate, and then by prep LCMS to give compound6-1. HPLC-MS (CI) m/z 604.1, 606.1 (MH⁺), t_(R)=26.98 (method 5),t_(R)=2.511 (method 4)

Example 73-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione

Step 7A: Preparation of 2-chloro-3-methoxybenzaldehyde 7a

To a suspension of 3-hydroxybenzaldehyde (20.12 g, 160 mmol) in HOAc (40mL) was added carefully tBuOCl (20 mL, 176 mmol) with stirring. Thereaction became a clear solution and strongly exothermic. It was allowedto cool and stirred for 16 hours, resulting in a white precipitate. Thesolid was filtered, washed with H₂O and dried to give2-chloro-3-hydroxybenzaldehyde (13.77 g, 55%), GCMS (EI) m/z 156, 158(M⁺).

To a solution of 2-chloro-3-hydroxybenzaldehyde (4.55 g, 29 mmol) in DMF(30 mL) was added K₂CO₃ (4.8 g, 34.9 mmol) followed by MeI (2.7 mL, 43.6mmol), and the mixture was stirred at room temperature for 16 hours.Following concentration in vacuo, the residual was taken up in ethylacetate, washed with H₂O, brine, dried over Na₂SO₄, and concentrated.Purification by column chromatography on silica gel with ethylacetate/hexanes 1/5 afforded 2-chloro-3-methoxybenzaldehyde 7a (4.68 g,94%) as a colorless oil, which solidified upon standing. GCMS (EI) m/z170, 172 (M⁺).

Step 7B: Preparation of2-chloro-1-methoxy-3-[2-(methylsulfanyl)-2-(methylsulfinyl)vinyl]benzene7b

To a solution of 2-chloro-3-methoxybenzaldehyde 7a (4.65 g, 27.3 mmol)and methyl(methylthio)methyl sulfoxide (4.3 mL, 43.9 mmol) in THF (25mL) was added a 40% methanolic solution of Triton B (6.2 mL, 13.6 mmol)and the resulting solution was refluxed for 16 hours. After THF wasremoved, the residue was taken up in ethyl acetate, washed with 1 N HCl,H₂O, and brine, then was dried over Na₂SO₄, and concentrated.Purification by column chromatography on silica gel with dichloromethaneafforded2-chloro-1-methoxy-3-[2-(methylsulfanyl)-2-(methylsulfinyl)vinyl]benzene7b (3.61 g, 48%) as a yellow oil. GCMS (EI) m/z 225 (M⁺-Cl-16), 210(M⁺-Cl-OMe).

Step 7C: Preparation of ethyl(2-chloro-3-methoxyphenyl)acetate 7c

To a solution of2-chloro-1-methoxy-3-[2-(methylsulfanyl)-2-(methylsulfinyl)vinyl]benzene7b (3.58 g, 12.9 mmol) in ethanol (20 mL) was added a 5 M ethanolicsolution of HCl (5.2 mL) and the resulting solution was refluxed for 3hours. After evaporation, the residue was purified by columnchromatography on silica gel with dichloromethane to affordethyl(2-chloro-3-methoxyphenyl)acetate 7c (2.78 g, 94%) as a yellow oil.GCMS (EI) m/z 228, 230 (M⁺).

Step 7D: Preparation of ethyl2-(2-chloro-3-methoxyphenyl)-3-(dimethylamino)acrylate 7d

A solution of ethyl(2-chloro-3-methoxyphenyl)acetate 7c (2.78 g, 12mmol) in DMFDMA (16 mL, 120 mmol) was refluxed for 16 hours. Afterevaporation, the residue was purified by column chromatography on silicagel with ethyl acetate/hexanes 1/2 to 1/1 to afford unreactedethyl(2-chloro-3-methoxyphenyl)acetate 7c (1.8 g, 65%) first, and thenethyl 2-(2-chloro-3-methoxyphenyl)-3-(dimethylamino)acrylate 7d (1.1 g,32%; 90% based on recovered starting material) as a yellow syrup. MS(CI) m/z 284.0, 286.0 (MH⁺).

Step 7E: Preparation of5-(2-chloro-3-methoxyphenyl)pyrimidine-2,4(1H,3H)-dione 7e

To a mixture of ethyl2-(2-chloro-3-methoxyphenyl)-3-(dimethylamino)acrylate 7d (1.7 g, 6mmol), urea (1.08 g, 18 mmol) and NaI (2.7 g, 18 mmol) in acetonitrile(20 mL) was added TMSCl (2.3 mL, 18 mmol). The resulting mixture wasrefluxed for 16 hours, cooled to room temperature, and 1.0 M NaOH (30mL) was added. The resultant solution was stirred for 20 hours, andacetonitrile was removed in vacuo. The aqueous solution was washed withether, cooled in ice bath, and neutralized with 1 N HCl (30 mL). Theprecipitate was filtered, washed with additional H₂O, and dried to give5-(2-chloro-3-methoxyphenyl)pyrimidine-2,4(1H,3H)-dione 7e (1.24 g, 82%)as a pale yellow solid. MS (CI) m/z 253.1, 255.1 (MH⁺).

Step 7F: Preparation of5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione7f

To a suspension of5-(2-chloro-3-methoxyphenyl)pyrimidine-2,4(1H,3H)-dione 7e (2.2 g, 8.7mmol) in acetonitrile (25 mL) was added bis(trimethylsilyl)acetamide(4.3 mL, 17.4 mmol), and the resulting solution was refluxed for 1.5hours. The mixture was cooled to room temperature,2-fluoro-3-trifluoromethylbenzyl bromide (2.7 g, 10.5 mmol) was added,and reflux was resumed for 16 hours. The reaction was quenched byaddition of MeOH (25 mL) and stirring for 2 hours. After concentration,the residue was purified by column chromatography on silica gel withethyl acetate/hexanes 1/1 to afford5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione7f (3.3 g, 88%) as a white solid. MS (CI) m/z 429.0, 431.0 (MH⁺).

Step 7G: Preparation of3-[2(R)-(tert-butoxycarbonylamino)-2-phenylethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione7g

A mixture of5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione7f (75 mg, 0.175 mmol), K₂CO₃ (72 mg, 0.525 mmol) andN-(t-butyloxycarbonyl)-D-α-phenylglycinol mesylate (0.11 g, 0.35 mmol)in DMF (2 mL) was heated at 75° C. for 16 hours. The reaction wasdiluted with ethyl acetate, washed with H₂O and brine, dried over Na₂SO₄and concentrated. The residue was purified by column chromatography onsilica gel with ethyl acetate/hexanes 2/3 to afford compound 7g (82 mg,72%) as a white solid. MS (CI) m/z 548.0, 550.0 (MH⁺-Boc).

Step 7H: Preparation of3-[2(R)-amino-2-phenylethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione7h

Compound 7g (2.7 g, 4.2 mmol) was dissolved in dichloromethane (10 mL),TFA (14 mL, 175 mmol) was added, and the mixture was stirred at roomtemperature for 4.5 hours. After concentration, the residue was taken upin DCM and saturated aqueous NaHCO₃ was added. The aq. layer wasextracted with DCM. Combined organic extracts were dried over Na₂SO₄ andconcentrated to give compound 7h (2.2 g, 96%). MS (CI) m/z 548.0, 550.0(MH⁺).

3-[2(R)-amino-2-phenylethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]pyrimidine-2,4(1H,3H)-dione7h.1 was prepared by substitution of the appropriate starting materialusing the procedures provided above.

Step 7I: Preparation of3-[2(R)-{ethoxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione7i

To a solution of compound 7h (2.0 g, 3.65 mmol) in DMF (8 mL) was addedNa₂CO₃ (0.47 g, 4.38 mmol) followed by ethyl 4-bromobutyrate (0.83 mL,5.48 mmol). The mixture was heated at 95° C. for 1.5 hours, cooled toroom temperature, and partitioned between ethyl acetate and H₂O. Theorganic layer was washed with brine, dried over Na₂SO₄ and concentrated.The residue was purified by column chromatography on silica gel withethyl acetate/hexanes/triethylamine 500/500/5 to afford compound 7i(1.29 g) as a white solid. MS (CI) m/z 662.2, 664.2 (MH⁺)

Step 7J: Preparation of3-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione7-1

To compound 7i (0.7 g, 1.06 mmol) was added THF (6 mL) and H₂O (6 mL)followed by NaOH (0.17 g, 4.24 mmol). The mixture was stirred at 50° C.for 16 hours. THF was removed in vacuo, the aq. solution was washed withether, and cooled at 0° C. Neutralization with 5% aq. citric acid (6.0mL, 4.7 mmol)) gave a precipitate, which was collected and furtherpurified by column chromatography on silica gel withMeOH/DCM/triethylamine 8/100/2 to afford compound 7-1 (0.56 g, 84%) as awhite solid. HPLC-MS (CI) m/z 634.2, 636.2 (MH⁺), t_(R)=24.925, (method5)

Example 83-[2(R)-{hydroxycarbonylpropyl-amino}-2-(isobutyl)ethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione

Step 8A: Preparation of3-[2(R)-{tert-butoxycarbonyl-amino}-2-(isobutyl)ethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione8a

To a solution of N-(t-butyloxycarbonyl)-D-α-leucinol (1.21 g, 5.57 mmol)in pyridine (6 mL) was added tosyl chloride (1.6 g, 8.35 mmol). Thereaction mixture was stirred at room temperature for 3 hours, dilutedwith EtOAc, and washed sequentially with 1 N HCl, H₂O, sat'd aq. NaHCO₃and brine. The organic layer was dried over Na₂SO₄, concentrated andpurified by column chromatography on silica gel with ethylacetate/hexanes 1/3 to afford[3-methyl-1-[[[(4-methylphenyl)sulfonyl]oxy]methyl]butyl]-1,1-dimethylethylcarbamic ester (1.66 g, 80%), MS (CI) m/z 272.2 (MH⁺-Boc).

A mixture of5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione7f (56 mg, 0.13 mmol), K₂CO₃ (754 mg, 0.39 mmol) and[3-methyl-1-[[[(4-methylphenyl)sulfonyl]oxy]methyl]butyl]-1,1-dimethylethylcarbamic ester (97 mg, 0.26 mmol) in DMF (2 mL) was heated at 95° C. for16 hours. The reaction was diluted with ethyl acetate, washed with H₂Oand brine, dried over Na₂SO₄ and concentrated. The residue was purifiedby column chromatography on silica gel with ethyl acetate/hexanes 1/1 toafford recovered[3-methyl-1-[[[(4-methylphenyl)sulfonyl]oxy]methyl]butyl]-1,1-dimethylethylcarbamic ester (30 mg, 54%) and compound 8a (30 mg, 37%), MS (CI) m/z528.0, 530.0 (MH⁺-Boc).

Step 8B: Preparation of3-[2(R)-amino-2-(isobutyl)ethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione8b

To a solution of compound 8a (30 mg, 0.048 mmol) in DCM (1 mL) was addedTFA (0.1 mL, 1.3 mmol) and stirred at room temperature for 1.5 hours.After concentration, the residue was taken up in DCM and sat'd aq.NaHCO₃ was added. The aq. layer was extracted with DCM. Combined organicextracts were dried over Na₂SO₄ and concentrated to give compound 8b. MS(CI) m/z 528.0, 530.0 (MH⁺).

Step 8C: Preparation of3-[2(R)-{ethoxycarbonylpropyl-amino}-2-(isobutyl)ethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione8c

To a solution of compound 8b (25 mg, 0.048 mmol) in DMF (1 mL) was addedK₂CO₃ (21 mg, 0.15 mmol) followed by ethyl 4-bromobutyrate (0.015 mL,0.1 mmol). The mixture was heated at 95° C. for 16 hours, cooled to roomtemperature, and partitioned between ethyl acetate and H₂O. The organiclayer was washed with brine, dried over Na₂SO₄ and concentrated. Theresidue was purified by column chromatography on silica gel with ethylacetate/hexanes/triethylamine 500/500/5 to afford compound 8c. MS (CI)m/z 642.2, 644.2 (MH⁺).

Step 8D: Preparation of3-[2(R)-{hydroxycarbonylpropyl-amino}-2-(isobutyl)ethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]pyrimidine-2,4(1H,3H)-dione8-1

To compound 8c (10 mg, 0.016 mmol) was added THF (0.3 mL) and H₂O (0.3mL) followed by NaOH (6.4 mg, 0.16 mmol). The mixture was stirred at 50°C. for 16 hours, and purified by prep LCMS to give compound 8-1. MS (CI)m/z 614.1, 616.1 (MH⁺), t_(R)=6.550 min (method 6)

Example 93-[2(R)-{2-[1-(5-tetrazoyl)propyl]-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]pyrimidine-2,4(1H,3H)-dione

Step 9A: Preparation of3-[2(R)-{2-[3-cyanopropyl]-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]pyrimidine-2,4(1H,3H)-dione9a

A solution of 7h.1 (2.59 g, 5 mmol) in CH₃CN (25 mL) was addeddiisopropylethyl amine (2.61 mL, 15 mmol), followed by the addition of4-bromobutyronitrile (2.22 g, 15 mmol). The reaction mixture wasrefluxed for 16 hours. Volatiles were evaporated and the residue waspurified by flash chromatography (silica, 4% MeOH/CH₂Cl₂) to givecompound 9a (2.62 g, 95.5%). MS (CI) m/z 549.1 (MH⁺).

Step 9B: Preparation of3-[2(R)-{2-[5-tetrazoylpropyl]-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]pyrimidine-2,4(1H,3H)-dione9-1

A solution of 9a (274 mg, 0.5 mmol) in DMF (5 mL) was added sodium azide(97 mg, 1.5 mmol) and ammonium chloride (120 mg, 2.25 mmol). Thereaction mixture was heated at 110° C. for 12 hours. The mixture wascooled, partitioned between EtOAc and saturated NaHCO₃/water, washedwith brine, dried (sodium sulfate), and evaporated. The residue waspurified by flash chromatography (silica, 6% MeOH/CH₂Cl₂) to givecompound 9-1 (52 mg, 17.6%). HPLC-MS (CI) m/z 592.3 (MH⁺), t_(R)=2.150,(method 4)

Example 103-[2(R)-amino-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-methylsulfonylbenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione

Step 10A: Preparation of3-[2(R)-tert-butoxycarbonylamino-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2,6-difluorobenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione10a

To a solution of compound 1f.1 (28 g, 56 mmol) in dichloromethane (200mL) was added a solution of di-tert-butyldicarbonate (12 g, 56 mmol) indichloromethane (100 mL) dropwise through an addition funnel. Thereaction mixture was stirred at room temperature for 2 hours. Thereaction mixture was concentrated by vacuum to yield the desired product10a as a light yellow solid (33 g, 56 mmol, 100%). HPLC-MS (CI)m/z=496.1 (M+H⁺-Boc), t_(R)=3.052 (method 4)

Step 10B: Preparation of3-[2(R)-tert-butoxycarbonylamino-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-methylthiobenzyl]-6-methyl-pyrimidine-2.4(1H,3H)-dione10b

To a solution of compound 10a (33 g, 56 mmol) in dry DMSO (100 mL) wasadded sodium thiomethoxide (4.0 g, 56 mmol) under nitrogen. The reactionmixture was heated to 100° C. under nitrogen for 1 hour. Another 0.28eq. of sodium thiomethoxide (1.1 g, 16 mmol) was added, and the reactionmixture was heated to 100° C. under nitrogen for 1 hour. The reactionmixture was cooled and partitioned between ethyl ether and water. Theorganic layer was washed with saturated aqueous sodium bicarbonatesolution and brine, dried with sodium sulfate, filtered andconcentrated. The crude product was purified with a flash chromatographyon silica gel eluted with 50% ethyl acetate in hexane to yield compound10b as a pale yellow solid (27 g, 44 mmol, 78%). HPLC-MS (CI) m/z=524.1(M+H⁺-Boc), t_(R)=3.134 (method 4). ¹H NMR (CDCl₃): 1.38 (s, 9H), 2.07(s, 3H), 2.51 (s, 3H), 3.90 (s, 3H), 4.07-4.13 (m, 1H), 4.29-4.39 (m,1H), 5.30-5.53 (m, 2H), 5.79-5.85 (m, 1H), 6.80-6.91 (m, 2H), 6.70 (dd,1H), 7.06-7.15 (m, 2H), 7.22-7.41 (m, 6H).

Step 10C: Preparation of3-[2(R)-tert-butoxycarbonylamino-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-methylsulfonylbenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione10c

To a solution of compound 10b (27 g, 44 mmol) in anhydrousdichloromethane (400 mL) was added 3-chloroperoxybenzoic acid (mCPBA, 30g, 180 mmol). The reaction mixture was stirred at room temperatureovernight. The reaction mixture was partitioned between dichloromethaneand water. The organic layer was washed with saturated aqueous sodiumbicarbonate solution and brine, dried with sodium sulfate, filtered andconcentrated. The crude product was purified with a by chromatography onsilica gel eluting with 50% ethyl acetate in hexane to yield the desiredproduct compound 10c as a pale yellow solid (15 g, 24 mmol, 53%).HPLC-MS (CI) m/z=556.0 (M+H⁺-Boc), t_(R)=2.941 (method 4). ¹H NMR(CDCl₃): 1.38 (s, 9H), 2.27 (brs, 3H), 3.48 (s, 3H), 3.92 (s, 3H),4.01-4.15 (m, 1H), 4.24-4.40 (m, 1H), 4.95-5.05 (m, 1H), 5.58-5.68 (m,2H), 6.85-6.91 (dd, 1H), 7.02 (dd, 1H), 7.14 (d, J=7.6 Hz, 1H),7.19-7.55 (m, 7H), 7.97 (d, J=7.6 Hz, 1H).

Step 10D: Preparation of3-[2(R)-amino-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-methylsulfonylbenzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione10-1

To a solution of compound 10c (10 g, 15 mmol) in anhydrousdichloromethane (60 mL) was added trifluroacetic acid (TFA, 16 mL). Thereaction mixture was stirred at room temperature for 4 hours. Thereaction mixture was concentrated, and partitioned between ethyl acetateand diluted aqueous NaOH solution. The organic layer was washed withsaturated aqueous sodium bicarbonate solution and brine, dried withsodium sulfate, filtered and concentrated to yield 10-1 as a tan solid(8.0 g, 14 mmol, 94%). HPLC-MS (CI) m/z=556.2 (M+H⁺), t_(R)=2.354(method 4). ¹H NMR (CDCl₃): 2.25 (s, 3H), 3.42 (s, 1.5H), 3.43 (s,1.5H), 3.91 (s, 1.5H), 3.92 (s, 1.5H), 3.98-4.22 (m, 2H), 4.33-4.38 (m,1H), 5.60 (brs, 2H), 6.80-6.89 (m, 1H), 6.97-7.03 (m, 1H), 7.11-7.17 (m,1H), 7.22-7.37 (m, 6H), 7.46-7.54 (m, 1H), 7.95 (dd, 1H).

Example 113-[2(R)-{2-[1-(5-tetrazoyl)propyl]-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-methylsulfonylbenzyl]-6-methylpyrimidine-2,4(1H,3H)-dione

Step 11A: Preparation of5,5′-[2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diylbis(ethane-2,1-diyl)]bis-1H-tetrazole

3,9-bis(2-Cyanoethyl)-2,4,8,10-tetraoxaspiro[5.5]undecane (5.38 g, 20mmol), azidotrimethylsilane (10.6 mL, 80 mmol), and dibutyl tin oxide(2.48 g, 4 mmol) were suspended in 40 mL toluene and 40 mL dioxane andheated at reflux for 18 hours. The reaction was cooled to roomtemperature and diluted with 100 mL hexane. The solid precipitate wascollected, washed with hexane (2×30 mL) and dried in air. The solid wassuspended in 100 mL 5% sodium carbonate solution, enough ethyl acetatewas added to dissolve most of the solid, and the mixture was stirred for1 hour. The layers were separated, the aqueous layer was washed withethyl acetate (2×100 mL), and the organic layers were back extractedwith 5% sodium carbonate (1×50 mL). The aqueous layers were combined,acidified to pH 7 with concentrated hydrochloric acid, filtered throughCelite, and acidified to pH 3. The solid was collected, washed withwater (2×50 mL) and acetone (2×50 mL) and dried under vacuum to give5,5′-[2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diylbis(ethane-2,1-diyl)]bis-1H-tetrazole11a (4.71 g, 67%). ¹H NMR (300 MHz, DMSO-d₆) δ 4.56 (t, 2H, J=5 Hz),4.28 (dd, 2H, J=9, 2 Hz), 3.58 (d, 2H, J=11 Hz), 3.57 (dd, 2H, J=11.2Hz), 3.36 (d, 2H, J=11 Hz), 2.94 (t, 4H, J=7.5 Hz), 1.97 (dt, 4H, J=8, 4Hz).

Step 11B: Preparation of3-[2(R)-[2-[1-(5-tetrazoyl)propyl]-amino]-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-methylsulfonylbenzyl]-6-methylpyrimidine-2,4(1H,3H)-dione11-1

A 25 mg sample of5,5′-[2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diylbis(ethane-2,1-diyl)]bis-1H-tetrazole1a (70 μmol) and p-toluenesulfonic acid (20 mg, 100 μmol) were suspendedin 1 mL of water and heated at 80° C. for 18 hours. The solution wascooled and added to compound 10-1 (29 mg, 50 μmol) dissolved in 1 mLethanol and 17 uL triethylamine (100 μmol).) Borane-pyridine complex (24μL, 240 μmol) was then added and the mixture stirred 0.25 hours untilbubbling ceased. The volatiles were removed and the residue taken up in2 mL ethyl acetate and washed with water (1×0.5 mL). The ethyl acetatelayer was evaporated and purified by preparative LC/MS to give 11-1 (5mg, 12% yield).

The following compounds were synthesized according to the aboveprocedure.

t_(R) No. R_(1a) R_(1b) R_(2a) R₃ MW Mass (Method #) 11-1 F OMe SO₂MeCH₃ 665.7 666.2 20.92 (5) 11-2 Cl H CF₃ H 628.0 628.2 27.34 (5) 11-3 FOMe F CH₃ 605.6 606.2 24.19 (5) 11-4 F OMe CF₃ CH₃ 655.6 656.2 2.540 (4)

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

1. A compound having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein: R_(1a), R_(1b) and R_(1c) are the same or different andindependently hydrogen, halogen, C₁₋₄alkyl, hydroxy or alkoxy, or R_(1a)and R_(1b) taken together form —OCH₂O— or —OCH₂CH₂—; R_(2a) and R_(2b)are the same or different and independently hydrogen, halogen,trifluoromethyl, cyano or —SO₂CH₃; R₃ is hydrogen or methyl; R₄ isphenyl or C₃₋₇alkyl; R₅ is hydrogen or C₁₋₄alkyl; R₆ is —COOH or an acidisostere; and X is C₁₋₆alkanediyl optionally substituted with from 1 to3 C₁₋₆alkyl groups.
 2. The compound of claim 1 wherein R_(1a) ishalogen.
 3. The compound of claim 1 wherein R_(1b) is alkoxy.
 4. Thecompound of claim 3 wherein R_(1b) is methoxy.
 5. The compound of claim1 wherein R_(1c) is halogen.
 6. The compound of claim 5 wherein R_(1c)is fluoro or chloro.
 7. The compound of claim 1 wherein R_(2a) ishalogen.
 8. The compound of claim 1 wherein R_(2b) is hydrogen, halogenor —SO₂CH₃.
 9. The compound of claim 1 wherein R₃ is hydrogen.
 10. Thecompound of claim 1 wherein R₃ is methyl.
 11. The compound of claim 1wherein R₄ is phenyl.
 12. The compound of claim 1 wherein R₄ isC₃₋₇alkyl.
 13. The compound of claim 12 wherein C₃₋₇alkyl is cyclopentylor cyclohexyl.
 14. The compound of claim 1 wherein R₅ is H or methyl.15. The compound of claim 1 wherein R₆ is —COOH. 16 The compound ofclaim 1 wherein R₆ is an acid isostere.
 17. The compound of claim 16wherein the acid isostere is tetrazol-5-yl.
 18. The compound of claim 1wherein X is a straight chain C₁₋₆alkanediyl.
 19. The compound of claim18 wherein the straight chain C₁₋₆alkanediyl is —CH₂CH₂CH₂—.
 20. Thecompound of claim 19 wherein R₄ is phenyl.
 21. The compound of claim 20wherein R_(1a) and R_(2a) are halogen.
 22. The compound of claim 21wherein R₃ is methyl.
 23. The compound of claim 21 wherein R₃ ishydrogen.
 24. The compound of claim 1 wherein X is a branchedC₁₋₆alkanediyl.
 25. The compound of claim 1 wherein the compound is3-[2(R){hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione,3-[2(R){hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(3-isopropylphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione,3-[2(R){hydroxycarbonylpropyl-amino}-2-(cyclohexyl)ethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-6-methyl-pyrimidine-2,4(1H,3H)-dione,or3-[2(R){2-[1-(5-tetrazoyl)propyl]-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-methylsulfonylbenzyl]-6-methylpyrimidine-2,4(1H,3H)-dione.26. The compound of claim 1 wherein the compound is3-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione,3-[2(R){hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-fluoro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione,3-[2(R)-{hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chloro-3-methylphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione,3-[2(R){hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chlorophenyl)-1-[2-fluoro-6-(methylsulfonyl)benzyl]-pyrimidine-2,4(1H,3H)-dione,3-[2(R){hydroxycarbonylpropyl-amino}-2-phenylethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione,or3-[2(R){hydroxycarbonylpropyl-amino}-2-(isobutyl)ethyl]-5-(2-chloro-3-methoxyphenyl)-1-[2-fluoro-6-(trifluoromethyl)benzyl]-pyrimidine-2,4(1H,3H)-dione.27. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier or diluent.
 28. A method forantagonizing gonadotropin-releasing hormone in a subject in needthereof, comprising administering to the subject an effective amount ofa compound of claim
 1. 29. A method for treating a sex-hormone relatedcondition of a subject in need thereof, comprising administering to thesubject an effective amount of the pharmaceutical composition of claim27.
 30. The method of claim 29 wherein the sex-hormone related conditionis cancer, benign prostatic hypertrophy or myoma of the uterus.
 31. Themethod of claim 30 wherein the cancer is prostatic cancer, uterinecancer, breast cancer or pituitary gonadotroph adenomas.
 32. The methodof claim 31 wherein the cancer is prostatic cancer.
 33. The method ofclaim 29 wherein the sex-hormone related condition is endometriosis,polycystic ovarian disease, uterine fibroids or precocious puberty. 34.The method of claim 33 wherein the sex-hormone related condition isendometriosis.
 35. The method of claim 29 wherein the sex-hormonerelated condition is uterine fibroids.
 36. A method for treatinginfertility of a subject in need thereof, comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 27. 37. A method for treating lupus erythematosis, irritable bowelsyndrome, premenstrual syndrome, hirsutism, short stature or sleepdisorders of a subject in need thereof, comprising administering to thesubject an effective amount of the pharmaceutical composition of claim27.